Download Molecular organization of the cell wall of Candida albicans and its

MINIREVIEW
Molecular organization of the cell wall of Candida albicans and its
relation to pathogenicity
José Ruiz-Herrera1, M. Victoria Elorza2, Eulogio Valentı́n2 & Rafael Sentandreu2
1
Departamento de Ingenierı́a Genética, Unidad Irapuato, Centro de Investigación y de Estudios Avanzados del IPN, Irapuato, Mexico; and
Departament de Microbiologı́a i Ecologı́a, Facultat de Farmacia, Universitat de València, Burjassot, Spain
2
Correspondence: José Ruiz-Herrera,
Departamento de Ingenierı́a Genética,
Unidad Irapuato, Centro de Investigación y de
Estudios Avanzados del IPN, Apartado Postal
629, 36500 Irapuato, Gto, Mexico. Tel.: 152
462 6239600; fax: 152 462 6245849; email: [email protected]
Received 22 June 2005; revised 18 August
2005; accepted 18 August 2005.
First published online 8 December 2005.
doi:10.1111/j.1567-1364.2005.00017.x
Editor: Lex Scheffers
Keywords
Candida albicans; cell wall; pathogenesis;
glucans; chitin; glycoproteins.
Abstract
Candida albicans is one of the most important opportunistic pathogenic fungi.
Weakening of the defense mechanisms of the host, and the ability of the
microorganism to adapt to the environment prevailing in the host tissues, turn the
fungus from a rather harmless saprophyte into an aggressive pathogen. The disease,
candidiasis, ranges from light superficial infections to deep processes that endanger
the life of the patient. In the establishment of the pathogenic process, the cell wall of
C. albicans (as in other pathogenic fungi) plays an important role. It is the outer
structure that protects the fungus from the host defense mechanisms and initiates
the direct contact with the host cells by adhering to their surface. The wall also
contains important antigens and other compounds that affect the homeostatic
equilibrium of the host in favor of the parasite. In this review, we discuss our present
knowledge of the structure of the cell wall of C. albicans, the synthesis of its different
components, and the mechanisms involved in their organization to give rise to a
coherent composite. Furthermore, special emphasis has been placed on two further
aspects: how the composition and structure of C. albicans cell wall compare with
those from other fungi, and establishing the role of some specific wall components
in pathogenesis. From the data presented here, it becomes clear that the composition, structure and synthesis of the cell wall of C. albicans display both subtle and
important differences with the wall of different saprophytic fungi, and that some of
these differences are of utmost importance for its pathogenic behavior.
Introductory remarks
Invasive fungal infections are important causes of
morbidity and mortality, mainly in immunodepressed and
hospitalized patients. Among these, the infectious processes
caused by Candida albicans have acquired an increasing
importance. Different virulence factors are involved in
C. albicans pathogenicity, but the role of the cell wall in the
pathogenesis of this fungus cannot be overestimated. The
wall is the structure that: (1) first comes into contact
with host cells; (2) carries important antigenic determinants
of the fungus; (3) is responsible for the adherence
of the pathogen; and (4) establishes a cross-talk with the
host, which depends on what has been referred to as
the ‘glycan code’, which includes modifications in the
chemical composition and linkages of the cell wall
polysaccharides.
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From these interactions, the outcome will be the development of a pathogenic state, or the mounting of a resistant
reaction by the host (for a discussion, see Poulain & Jouault,
2004). As in fungi in general, the cell wall of C. albicans is a
coherent structure, made by the ordered arrangement of its
different components. Some of these are linked by covalent
bonds, whereas others are retained in the wall by hydrogen
bonds, salt-type associations, hydrophilic or hydrophobic
interactions. This composite provides protection to the cell
against physical, chemical and biological aggression, and is
responsible for its morphology.
Microscopic observation of thin sections of fungal cells or
isolated walls has revealed the existence of several layers in
the wall, distinguished by their electron density in electron
micrographs. Depending on the method of analysis, the wall
of C. albicans appears to contain from four to eight layers
(Poulain et al., 1978) (see Fig. 1a).
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Molecular organization of the cell wall of Candida albicans
Mannoproteins are enriched in the outer surface of
the C. albicans wall, including the bud scar (Horisberger &
Clerc, 1988). Once considered to be absent in the inner
layers, their presence has been demonstrated by different
techniques, including labeling with ConA or antibodies
(Marcilla et al., 1991; Kapteyn et al., 2000). In different
fungi, including C. albicans, bona fide wall proteins (see
below) are bound to the b-glucan/chitin inner layer through
the lateral chains of b-1,6-glucan or, to a lesser extent, to b1,3-glucan. The structural polysaccharides accumulate in
the innermost layer of the cell wall, which appears to be less
electron-dense. After extensive treatment of C. albicans
blastoconidia (yeast-form) with a-mannosidase and alkaline
phosphatase, b-1,6-glucan, but not b-1,3-glucan or chitin,
becomes accessible to labeling (see a schematic representation in Fig. 1).
Labeling with positively charged polycationic colloidal
gold–chitosan complexes, and observation by electron mi-
croscopy, reveals the presence of anionic complexes on the
surface of blastoconidia, germ tubes and hyphae (Horisberger & Clerc, 1988). Apparently, these anionic radicals are
homogeneously dispersed over the surface of older blastoconidia, except at the bud scars. Their presence on the
surface of emerging buds and young cells depends on the
growth conditions. In hyphal cells, surface anionic radicals
are more abundant, especially at the apex, and are associated
with a fuzzy coat that appears external to the wall and seems
to play a role in pathogenicity, phagocytosis and adherence.
It is feasible that those anionic compounds correspond to
glycoproteins containing abundant phosphodiester linkages
that confer a negative charge to the cells at physiological pH,
and provide hydrophilic or hydrophobic properties to the
cell surface, as occurs in Saccharomyces cerevisiae (Jigami &
Odani, 1999). Accordingly, hydrophobic properties of C.
albicans cells, which are important for virulence, may
depend on the polysaccharides located in the outer part of
Fig. 1. Structure and schematic representation of the architecture of Candida albicans cell wall. (a) Electron micrograph of a median cell section. The
electron transparent inner layer of the wall (thin black and white arrow) is made mainly of polysaccharides (b-glucans and chitin) and small amounts of
proteins. The electron-dense outer layer (thick black arrow) is built mostly of different types of mannoproteins. (b) Scheme of the cell wall. b-1,3/1,6Glucan chains are linked by covalent bonds to chitin microfibrils and, together with some proteins, give rise to a basic composite (A). The outer surface
of this composite (B) is enriched in different types of proteins which are anchored by either noncovalent bonds or by an assortment of covalent linkages.
(c) Schematic representation of the molecular organization of the cell wall. Cell wall proteins are attached mainly to short chains of b-1,6-glucan, to
chitin via b-1,6-glucan, or directly to chitin. GPI, glycosyl phosphatidylinositol proteins; ASL, proteins bound through alkali-sensitive bonds; RAE,
proteins bound by disulfide bridges. (For additional information, see text.)
FEMS Yeast Res 6 (2006) 14–29
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the wall, specifically the phosphodiester-linked, acid-labile
b-1,2-mannan, which determines the serotype classification.
It has been found that the acid-labile oligomannosides from
hydrophobic cells are longer and potentially present in
greater abundance than those from hydrophilic cells, and
that switching in the hydrophobic status of C. albicans
strains depends on a modification of the chain length of
these acid-labile b-1,2-oligomannosides. Surprisingly, however, mutation of the gene coding for the enzyme responsible for mannosyl phosphate transfer, and hence for the
attachment of b-1,2-mannose oligosaccharides to the acidlabile N-mannan side chains of wall glycoproteins, did not
affect morphogenesis, virulence or recognition of the fungus
by macrophages (Hobson et al., 2004). On the other hand,
mutation of MNT1 and MNT2 genes, which encode partially redundant a-1,2-mannosyltransferases that catalyze
the addition of the second and third mannose residues in
the O-linked mannose pentamers, resulted in truncation of
O-mannan, reduction in the level of mannosyltransferase
activity, reduced virulence and reduced adherence, indicating a more important role for O-mannan a-chains in
adhesion to host surfaces (Munro, 2005). The capacity to
form biofilms (structured microbial communities with high
levels of drug resistance) is also dependent on the hydrophobic properties of the cell surface. This is an important
property that permits C. albicans to colonize host tissues,
implants and surgical material. Farnesol, a quorum-sensing
molecule that inhibits hyphal formation in C. albicans, has
been found to prevent biofilm formation by the fungus.
Using microarray analysis of farnesol-treated populations, a
series of gene products important for biofilm formation
were identified (Cao et al., 2005). These included, among
others, some hyphae-associated proteins, chitinases and
proteins associated with the hydrophobic properties of the
cells.
Cell wall lipids
Lipids constitute minor components of the cell wall of
Candida albicans, and of fungi in general. An interesting
lipid in the wall of C. albicans is phospholipomannan, which
reacts with antibodies specific to b-1,2-oligomannosides
(Mille et al., 2004). Phospholipomannan lacks glucosamine
and displays a distinct organization of the glycan chains.
Analysis by radiolabeling, methylation-methanolysis and
mass spectrometry evidenced a structure made of linear
chains of b-1,2-joined mannose residues with degrees of
polymerization varying from eight to 18 sugars, bound to
phosphoinositol ceramide. Phospholipomannan appears as
a novel type of eukaryotic inositol-tagged glycolipid, based
upon the absence of glucosamine and the organization of its
glycan chains. It has been suggested that this compound may
have some relevance in adhesion, protection and signaling
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J. Ruiz-Herrera et al.
in C. albicans. Interestingly, synthesis of phospholipomannan occurs by a unique mechanism diverging from the
usual pathway at the mannose-inositol-phospho-ceramide
(MIPC) step. Mutation of the MIT1 gene, encoding the
glucose degradation product (GDP)-MIPC mannose transferase, reduced virulence in a mouse model and resistance to
lysis by macrophages, indicating the importance of the
mannolipid in resistance and pathogenicity of C. albicans
(Mille et al., 2004).
Chitin
Chitin is a linear polysaccharide made of more than 2000
units of N-acetylglucosamine (2-acetamido-2-deoxy-D-glucose, GlcNAc) joined by b-1,4-linkages (Fig. 2; Table 1).
Chitin chains are associated in an antiparallel fashion
through hydrogen bonds to form microfibrils composed of
c. 20–400 chains. Owing to this crystalline arrangement,
chitin is one of the most insoluble natural products, and this
explains why its linkages to b-1,3-glucan form the basic cell
wall scaffold (see below) to which mannoproteins are
covalently associated.
Synthesis of chitin involves a transglycosylation reaction
of GlcNAc residues from the universal substrate UDP-Nacetylglucosamine to the growing chain of the polysaccharide. The reaction (catalyzed by ill-known enzymes called
chitin synthases, Chsps) requires a divalent metal, generally
Mg21, but does not involve a lipid or a high-energy
intermediate (Ruiz-Herrera & Ruiz-Medrano, 2004; RuizHerrera et al., 2004). Early studies revealed that S. cerevisiae
contained more than one Chsp, a property found later on to
be common to all fungi. This characteristic probably represents a mechanism that permits their adaptation to different
environments, and protects the cell in case of the loss of
one chitin synthase. Nevertheless, in some specific cases (see
below) a distribution of roles of the different enzymes
has been revealed. Comparison of fungal Chsps has led to
their classification into two divisions (1 and 2), the first
with three classes (I, II and III) and the second with two (IV
and V) (see review in Ruiz-Herrera & Ruiz-Medrano, 2004).
Chitin synthase is accumulated in the cytosol of fungi,
C. albicans included, in specialized microvesicles, chitosomes, responsible for the transfer of the enzyme from its
site of synthesis to its place of action (see review in RuizHerrera et al., 2004).
In agreement with the multigenic control of chitin
synthases in fungi, three genes encoding chitin synthases
(CHS genes) were originally described to exist in C. albicans.
One of them, CHS2, is preferentially expressed in the hyphal
form of the fungus. Nevertheless, its disruption did not
affect the dimorphic behavior of the fungus, its levels of wall
chitin or its virulence in a mouse model (Gow et al., 1994).
Expression of the three genes did not correlate with chitin
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Molecular organization of the cell wall of Candida albicans
Fig. 2. Chemical structures of chitin (a), b-1,3/1,6-glucan (b), a glycosyl phosphatidylinositol (GPI) protein (c) and CaPir1 (d). GPI proteins are rich in Ser/
Thr, indicating that the protein could be highly glycosylated, with one or more potential N-glycosylation sites (NST). The hydrophobic N- (signal peptide)
and C-terminal GPI attachment domains are also depicted. CaPir1 presents four cysteine residues in the C-terminal part of the protein (-C-66aa-C-16aaC-12aa-C-COOH) and has nine (IPF 15 363) or seven (IPF 19 968) internal repeats with the structure [-(A/K/Q)-Q-I-(S/T/G/N)-D-G-Q-I-Q-H-Q-T-]. In
addition, one potential NST in both IPF19 668 (amino acids 194–196) and IPF 15 363 (233–235) is present, and approximately 20% of the amino acids
are Ser or Thr, indicating that the protein could be highly glycosylated.
Table 1. Components of Candida albicans cell wall
Percentage of cell wall
(dry weight)
Blastospore Mycelium Chemical units
Linkages
Physical state
Chitin
2
6
N-acetylglu cosamine
b-1,4
Glucan
58–60
54–56
Glucose
b-1,3/1,6
Mannoproteins38–40
38–40
Amino acids, N-acetylglucosamine, Mannose
phosphorous
Peptide N-glycosydic
mainly a mannose
pyrophosphate
Crystalline
antiparallel
associated chains
Amorphous with
microcrystalline
segments
Amorphous
levels of the cell. CHS2 and CHS3 reached maximal expression 1–2 h after hyphal induction, but CHS1 gene expression
remained at low levels in both yeast and hyphae. Chs2p is
the most abundant enzyme measured in vitro, but Chs3p is
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Chemical
solubilization
Biological
degradation
–
Chitinase
Partial degradation
with acid
b-glucanase
Detergents, reducing Proteases
agents, HF-pyridine
Degradation with
alkali and acids
responsible for in vivo synthesis of most chitin in both yeast
and hyphae (Mio et al., 1996). Nevertheless, Chs1p was
found to be essential for cell integrity and virulence,
apparently being involved in septum formation (Mio et al.,
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1996). Null mutants form long chains without septa, and
eventually lyse. Mutants defective in Chs3p are less virulent
than the parental strain in a mouse model. By use of a pair of
primers corresponding to the CHS1 gene, it was possible
to amplify by PCR specific fragments of the homologous
CHS1 genes from the medically most important species of
Candida: C. albicans, Candida parapsilosis, Candida tropicalis and Candida (Torulopsis) glabrata. The technique offered
the possibility to correctly identify the Candida species
present in tissue or blood samples (Jordan, 1994). More
recently, by use of in silico analysis of the genome sequence, a
fourth CHS gene encoding a putative Chsp has been
identified in C. albicans. The gene (CaCHS8) encodes a class
I chitin synthase with similarities to Chs2p. Null chs8
homozygous mutants display normal growth rates, cellular
morphology and chitin content, but their chitin synthase
activity is reduced to 25%, and the cells were hypersensitive to Calcofluor white. A double homozygous
chs2chs8 mutant possesses less than 3% of the wild-type
Chsp activity in vitro but has normal growth rate and
morphology. Mutation of the C. albicans homolog of the S.
cerevisiae BNI4 gene, encoding the protein responsible for
tethering of Chs3p during budding, brought about a reduction in chitin levels and morphological alterations, but not
in the chitin ring that separates mother and daughter cell
during budding.
Chitin synthase has repeatedly been proposed to be an
adequate target for the control of mycoses, taking into
consideration the importance of chitin in the structure of
the fungal cell wall, and its absence in the host. Two
important families of inhibitors of chitin synthase have been
described: polyoxins and nikkomycins. Despite their high
activity in vitro, their results in vivo have been disappointing. Chemical modification, to obtain derivatives with an
in vivo antifungal activity, or isolation of novel compounds
appears to be a promising approach (Ruiz-Herrera & SanBlas, 2003).
Cell wall glucans
Candida albicans does not contain a-glucans. It only contains b-glucans, and these are the most abundant polysaccharides of the fungal cell wall in general. They are polymers
of glucose moieties joined by b-1,3 and/or b-1,6-glycosidic
linkages (Fig. 2; Table 1). b-1,3-Glucan chains possess a
helical or spiral backbone, existing as a single helical polymer strand or as a complex of three polymer chains (triple
helix) that is stabilized by extensive hydrogen bonding at the
C-2 hydroxyl residue. The triple helical form appears to be
the preferred and most common molecular conformation in
nature. Unbranched b-1,3-glucans have a microfibrillar
structure, as revealed by electron microscopy (Kreger &
Kopeck, 1975). Candida albicans cell walls contain both b2005 Federation of European Microbiological Societies
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J. Ruiz-Herrera et al.
1,3 and b-1,6-glucans, but no mixed intrachain b-1,3/1,6
linkages. An acid-soluble fraction was found to consist
mainly of highly branched b-1,6-glucan. The alkali-insoluble glucan from either yeast or hyphal cells contains 30–39%
b-1,3 and 43–53% b-1,6 linkages, whereas that from the
germ tubes has these proportions reversed: 67% b-1,3 and
14% b-1,6 linkages. Analysis by high-resolution, solutionstate proton nuclear magnetic resonance spectroscopy
(NMR) of glucans isolated from yeast or hyphal forms of
C. albicans demonstrated that they were different from S.
cerevisiae glucans in side-chain branching and reducing
termini. Glucans from the yeast-like form have a mean Mr
of over 106 Da, whereas the Mr of the mycelial glucan is
slightly higher. The existence of a covalent bond between b1,6-glucan and chitin through a glycosidic linkage at position 1 of glucose and 6 of N-acetylglucosamine was demonstrated in C. albicans (Surarit et al., 1988). Glucans and
complexes of b-glucans and mannans are released by C.
albicans in synthetic medium, and apparently also into the
blood of infected patients. These compounds are toxic and
may induce anaphylactic shock and coronary arteritis in
murine models. In addition, C. albicans glucans suppress
monocyte functions directly, and T-cell function indirectly,
suggesting that they may play a role in the development of
candidiasis (Nakagawa et al., 2003). On the other hand, antib-glucan antibodies participate in the immune response as a
result of their capacity to recognize pathogenic fungi (Ishibashi et al., 2005). Recent evidence has confirmed the
existence of glucan-specific receptors on cells outside the
immune system. This aspect is important, because it is
known that hormones and cytokines may act as regulatory
messengers between the neuroendocrine and immune systems. In this way, the innate immune system identifies
infectious agents by means of pattern-recognition receptors.
These receptors recognize pathogen-specific macromolecules called ‘pathogen-associated molecular patterns’. Fungal cell wall glucans nonspecifically stimulate various aspects
of innate immunity via interaction with membrane receptors on immune-competent cells. Breuel et al. (2004) have
hypothesized that glucans may directly interact with pituitary cells as an early signaling event in fungal infections, and
evidence was obtained that pituitary cells directly recognized
and responded to fungal cell wall glucans resulting in an
increase in the expression of TLR4 and CD14 genes. Additionally, glucans stimulated secretion of prolactin, a hormone that plays an important role in the response to
infection. It has been suggested repeatedly that the hyphal
form of C. albicans is the invasive stage of the fungus. In this
regard, a possible relationship between C. albicans morphogenesis and Dectin-1 deserves attention. Dectin-1 is a
receptor that binds b-glucans and is important for phagocytosis of fungi by macrophages. The receptor also collaborates with Toll-like receptors for inflammatory activation of
FEMS Yeast Res 6 (2006) 14–29
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Molecular organization of the cell wall of Candida albicans
phagocytes by fungi. It has been observed that glucans
remain protected from the receptor by other wall components, except during budding, when scar glucans become
accessible to binding. On the other hand, hyphae, which lack
a budding mechanism, escape from the receptor, and can
colonize the host tissues more effectively (Gantner et al.,
2005). Synthesis of b-glucans is a complex reaction involving several enzymes located at different cell compartments.
Chain growth of b-1,3-glucan is the best-known process. It
involves a transglycosylation reaction of glucosyl residues
from the universal donor UDP-glucose (UDPG) to the
growing polysaccharide chain (for a review, see Ruiz-Herrera et al., 2004). The reaction is stimulated by guanine
triphosphate (GTP) and does not involve a lipid intermediate. The final product obtained in vitro may be huge in size,
with an Mr of over 20 106 kDa, and with a microfibrillar
structure. Studies carried out with C. albicans and S.
cerevisiae provided evidence that initiation of synthesis of
the polymer requires a protein acceptor (Ruiz-Herrera et al.,
2004).
Identification of the catalytic b-1,3-transglycosidase moiety was achieved in Neurospora crassa by labeling a highly
purified sample with the photosensitive UDPG analog 5azido-(b-[32P]-UDPG). The results led to the unequivocal
identification of one of the labeled peptides as b-1,3-glucan
synthase (Schimoler-O’Rourke et al., 2003). Genes encoding
the enzyme, named FKS, have been isolated from different
fungi, including C. albicans, where three FKS homologs with
high similarity to yeast FKS1 were described: CaGSC1,
CaGSL1 and CaGSL2 (Mio et al., 1997). The main activity
appears to result from Gsc1p (CaFks1p). Inability to inactivate both CaFKS1 alleles, the target for glucan synthesis
inhibitors, has been taken as evidence that CaFks1p is
essential. It is interesting to recall that Fks proteins lack the
motifs characteristic of b-glycosyl transferases: three Asp
residues and the QXXRW motif (Campbell et al., 1997),
associated with the catalytic activity, and the proposed UDP
glucose-binding site (R/K) XGG. All these data suggest that
b-1,3-glucan synthases constitute a different family of
glucosyltransferases, probably with a distinct phylogenetic
origin.
Activity of glucan synthase requires a small GTPase,
referred to as Rho1, that is also involved in other essential
functions of the cell, including wall integrity and cell
morphogenesis. Originally described in S. cerevisiae (Qadota
et al., 1996), the genes encoding the Rho1 protein homologs
in a range of other fungi have since been identified. Rho1
protein from C. albicans is 82.9% identical to S. cerevisiae
Rho1p and contains all the domains conserved among Rhotype GTPases from other organisms. Recombinant C. albicans Rho1p copurifies with the b-1,3-glucan synthase catalytic subunit, and can rescue inactivated b-1,3-glucan
synthase from C. albicans and S. cerevisiae membranes.
FEMS Yeast Res 6 (2006) 14–29
Rho1p has been found to be essential in C. albicans.
Accordingly, by use of mutants of the fungus that conditionally expressed Rho1p, it has been observed that depletion of the protein in either yeast or hyphal cells leads to
aggregation, death and lysis. Such mutants are also avirulent
in a mouse model (Smith et al., 2002).
b-1,3-Glucan synthases constitute the target of important
inhibitors of glucan synthesis, and therefore of wall growth.
Among these, the most important are echinocandins,
synthetically modified lipopeptides that were originally
derived from products synthesized by different fungi. These
include: aculeacin A (from Aspergillus aculeatus), echinocandin B (from Aspergillus rugulovalvus), pneumocandin B
(from Zalerion arboricola), enfumafungin (from a Hormonema-like fungus) and papulacandins (from Papularia
sphaerosperma) (for a review, see Denning, 2002). The
mutation responsible for spontaneous echinocandin resistance in C. albicans was pinpointed to CaFKS1, the structural gene encoding the most important glucan synthase of the
fungus (Mio et al., 1997a). Today, the clinical pharmacology
of different echinocandin derivatives for treatment of patients suffering from fungal invasive infections is an important topic of research.
Enzymes belonging to a family of transglycosidases classified into a new group of glycoside hydrolases, Family 72,
appear to be important for the structural organization of the
cell wall. These were originally described as being involved in
the pH-regulated morphogenesis of C. albicans. Later on,
they were found to play a role in b-1,6 and b-1,3-glucans
bonding in the wall of the fungus (Fonzi, 1999). The
corresponding enzymes – Phr1p and Phr2p from C. albicans, Phr1p and Phr2p from Candida dubliniensis, Gasp1 to
5 from S. cerevisiae, and Phr1 from Pneumocystis carinii –
belong to the glycosyl phosphatidylinositol (GPI) family of
wall proteins. They internally split b-1,3-glucan molecules
and transfer the newly generated fragments containing a
reducing end to the nonreducing end of other b-1,3-glucan
molecules to form a new b-1,3-linkage (Mouyna et al.,
2000). Mutants defective in the genes encoding these
proteins display abnormal morphologies and alterations in
cell wall properties and structure (Muhlschlegel & Fonzi,
1997).
Regarding synthesis of b-1,6-glucan, most of our knowledge comes from studies with S. cerevisiae. Because b-1,6glucan is the receptor for killer toxin K1, toxin (kre)resistant mutants became the ideal tool for the identification
of genes involved in its synthesis. By these procedures, at
least 10 genes involved in b-1,6-glucan biosynthesis were
identified in yeast: KRE1, KRE5, KRE6, KRE9, KRE11,
CNE1, CWH41/GLS1, KNH1, ROT2/GLS2 and SKN1 (reviewed in Shahinian & Bussey, 2000). Homologs of KRE1,
KRE5, KRE6, CNE1, CWH41/GLS1, ROT2/GLS2 and SKN1
have been identified in the C. albicans genome.
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J. Ruiz-Herrera et al.
Genetic and structural studies suggest that b-1,6-glucan
synthesis may occur at the cell surface, but several intracellular events involving the secretory pathway are crucial for
the synthesis of the polymer. Accordingly, several gene
products involved in the synthesis of b-1,6-glucan have an
intracellular location. Kre5 and Cwh41 proteins are located
in the endoplasmic reticulum, whereas Kre6 and Skn1 are
transmembrane Golgi proteins (Shahinian & Bussey, 2000).
The C. albicans homologs of several KRE genes have been
isolated, providing evidence that the mechanism of synthesis
of b-1,6-glucan occurs by similar mechanisms in both fungi.
The CaKRE1 gene was isolated by its ability to complement
a kre1 mutation in S. cerevisiae. The predicted protein
encoded by CaKRE1 is structurally similar to that encoded
by the yeast gene (Boone et al., 1991). kre5 Mutants of C.
albicans have reduced b 1,6-glucan levels and severe wall
defects and do not form hyphae on solid medium even when
serum is added, although they do it in the presence of
GlcNac; besides, they are avirulent (Herrero et al., 2004).
cDNAs of KRE6 and SKN1 from C. albicans have been
isolated (Mio et al., 1997b). In the yeast phase, KRE6
expression was higher than that of SKN1, but expression of
the latter increased at the onset of hyphal growth. Homozygous skn1 null mutants were not affected in the levels of b1,3 or b-1,6-glucan, whereas in heterozygous kre6 null
mutants the levels of b-1,6-glucan were reduced by more
than 80% without affecting the amount of b-1,3-glucan. It
was not possible to isolate the homozygous mutant, thus
suggesting the essential role of Kre6. This was confirmed by
inhibition of KRE6 expression through the use of the HEX1
promoter. Repressed cells exhibited a partial defect in cell
separation and increased susceptibility to Calcofluor white
(Smith et al., 2002).
Cell wall proteins
It is beyond discussion that identification of cell wall
proteins (CWPs) has advanced rapidly in recent years as a
result of the introduction of three novel methodological
approaches: (1) sequencing of the whole genomes of fungi;
(2) in silico analysis of the genomes with the help of
ingenious programs and algorithms; and (3) extremely
sensitive proteomic techniques of analysis (mainly mass
spectrometry). However, despite these advances, the definition of cell wall proteins has become an elusive matter
because different proteins normally considered to be of
cytoplasmic location have consistently been found joined
to the cell walls in different fungi, C. albicans included (see
below). Accordingly, our definition of bona fide wall-bound
proteins (Sentandreu et al., 2004) has included only those
proteins that have a secretory motif, are N- and/or Oglycosylated, and possess other specific characteristics, such
as a GPI-binding motif or specific inner repeats (see below;
Table 1 and 2). In C. albicans, these analyses have identified
hundreds of ORFs encoding putative wall proteins (De
Groot et al., 2003; Garcerá et al., 2003).
In contrast to these ‘true’ CWPs, mass spectrometric
analyses have disclosed the presence of some nonglycosylated proteins in the cell wall of C. albicans (Pitarch et al.,
2002), whose relevance and mechanism of retention remain
obscure. For these reasons, an operational criterion was
introduced for a systematic description to include true (see
Table 2. Classification and characteristics of cell wall proteins
Protein
Class
Subclass
Group
1
2
2a ‘True’ wall proteins
I
II
III
2b ‘Atypical’ wall
proteins
Characteristics
NCL-CWP. Proteins extractable by ionic detergents or chaotropic agents. Secretory domain (signal
peptide), Ser/Thr rich functional and structural domains (N- and/or O-glycosylation). Retained by
noncovalent bonds
Proteins solubilized after degradation of the structural polysaccharides, or by breakage of specific bonds
GPI-CWP. GPI proteins. Secretory domain (signal peptide). Structural domain (N- and/or Oglycosylation). GPI binding domain. Linked through b-1,6-glucan to b-1,3-glucan or chitin
ASL-CWP. Alkaline soluble (sensitive) wall proteins. Secretory domain (signal peptide). Ser/Thr rich
functional and/or functional structural domain (N- and/or O-glycosylation). Most have also internal
repeats. Four Cys residues in most terminal parts. Often processed by Kex2. Lack GPI-binding domain.
Linked to b-1,3-glucan by unknown linkages
RAE-CWP. Reducing agents and extractable wall proteins. Secretory domain (signal peptide). Ser/Thr
rich functional and/or structural domain (N- and/or O-glycosylation). Some with internal repeats.
Connected to other proteins through disulfide linkages
Do not possess the canonical signal peptide and other characteristics of the ‘true’ cell wall proteins and
have been previously identified as members of different cytoplasmic pathways. Unknown mechanism
of association to cell wall
NCL-CWP, non-covalently linked cell wall proteins; GPI-CWP, glycosylphosphatidylinositol cell wall proteins; RAE-CWP, reducing agents extractable cell
wall proteins.
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FEMS Yeast Res 6 (2006) 14–29
21
Molecular organization of the cell wall of Candida albicans
above) and nontypical CWPs (Table 2; Fig. 1) (for a review,
see Sentandreu et al., 2004). Accordingly, CWPs were
separated into two classes: class 1, proteins which can be
extracted by hot water, but mainly by ionic detergents or
chaotropic agents (NCL-CWP); and class 2, proteins resistant to this treatment, and solubilized only after digestion of
the structural polysaccharides, or by breakage of specific
bonds through which they are bound to wall polysaccharides. This second group of proteins was divided into two
subclasses. Proteins from subclass 2a are glycoproteins
covalently linked to the cell wall – in other words, ‘true’
CWPs. Proteins from subclass 2b (nontypical proteins) are
devoid of a carbohydrate moiety and are retained in the wall
by unknown mechanisms. Three different types of covalently bound glycoproteins have been described in subclass
2a. Group I includes proteins bound to b-1,6-glucans
through a GPI moiety (GPI-CWP) (De Nobel & Lipke,
1994; De Groot et al., 2005). Group II corresponds to Pir
proteins (proteins with internal repeats), characterized as
containing repetitive sequences (Toh-e et al., 1993) and
being highly O-glycosylated. Pir proteins are attached to
b-1,3-glucan by unknown alkali-sensitive bonds (possibly
O-glycosidic linkages) (ASL-CWP). Recently, some proteins
without internal repeats but bound through alkali-sensitive
bonds have been described (Castillo et al., 2003; De Groot
et al., 2005). Group III is formed by mannoproteins (RAECWP) retained by disulfide bridges, which are extracted by
treatment with reducing agents such as b-mercaptoethanol
(b-ME) or dithiothreitol (Moukadiri & Zueco, 2001).
GPI proteins
Glycosyl phosphatidylinositol proteins are rich in Ser and
Thr residues, and are highly O-glycosylated (Fig. 2; Table 2).
The structure linking the C-terminal end of GPI proteins to
the lipid moiety is identical in GPI anchors from all
organisms analyzed so far, namely protein-CO-NH-(CH2)PO4-Man-a-1,2-Man-a-1,6-Man-a-1,4-GlcN-a-1,6-inositol-PO4-lipid. The core contains branches of a-1,3- and a1,2-linked mannose units (Sipos et al., 1995). However, the
GPI anchors from various species differ widely with regard
to the side chains attached to this core structure, as well as to
the lipid moieties of the anchor. It was suggested (Frieman &
Cormack, 2003) that the amino acids immediately upstream
of the site of GPI anchor addition (the o site) serve as the
signal determining whether a GPI protein localizes to the
cell wall or to the plasma membrane. This signal consists of a
region of hydrophobic amino acids, followed by another
short region of more hydrophilic amino acids and a binding
site formed by three amino acid residues named o, o11 and
o12. Cleavage of the protein takes place between o and
o11, and the GPI anchor remains bound to the o amino
acid (Nuoffer et al., 1993). Transfer of the GPI moiety to the
FEMS Yeast Res 6 (2006) 14–29
protein, which takes place in the lumen of the endoplasmic
reticulum, is carried out by a transamidation reaction
involving cleavage of a carboxy terminal hydrophobic sequence, with the concomitant formation of an amide
linkage between the ethanolamine phosphate of the GPI
and the new carboxy-terminal amino acid (Udenfriend &
Kodukula, 1995). Transfer is catalysed by a GPI-transamidase. Assembly of S. cerevisiae GPIs includes the addition of
a fourth, side-branching mannose to the third mannose of
the core GPI glycan by the Smp3 mannosyltransferase. The
gene encoding this enzyme has been cloned in C. albicans,
and was found to be essential for growth, suggesting that C.
albicans utilizes the same pathway as S. cerevisiae for GPI
synthesis (Grimme et al., 2004). In cell wall proteins, the
above described anchor is trimmed, and only a part is
retained at their C-termini, which participate in the binding
of the proteins to b-1,6-glucan (Lipke & Ovalle, 1998).
Quantitatively, GPI proteins appear to be the most
important ones in C. albicans, where they account for about
88% of all covalently linked wall proteins. These proteins are
bound via b-1,6-glucan to b-1,3-glucan (90%), or to chitin
(about 10%) (Marcilla et al., 1991). In Dmnn9 and Dpmt1
mutants that are defective in protein N- or O-glycosylation,
respectively, the levels of GPI proteins bound to chitin were
increased (Kapteyn et al., 2000). As indicated above, in silico
analyses have disclosed the presence of a large number of
GPI proteins in Candida species. Using the big-ii predictor
for fungal systems, a total of 234 putative GPI proteins,
bound to either the plasma membrane or the cell wall, have
been identified in C. albicans (Eisenhaber et al., 2004). In
Candida glabrata, the number was lower, namely 106 (Weig
et al., 2004). It was calculated that 50 of these were adhesive
in nature, 11 were glycoside hydrolases, others had a
different enzymatic activity, and the rest were probably
structural proteins. Among the different GPI proteins identified in Candida spp., we may cite adhesins, such as Hwp1,
the agglutinin-like sequence (ALS) family of C. albicans, and
Epa1p of C. glabrata (Sundstrom, 2002). Some GPI proteins
are essential. Accordingly, double mutants affected in Dfg5p
and Dcw1p, two similar GPI proteins, are synthetically lethal
(Spreghini et al., 2003). In C. albicans, a proteomic analysis
of cell wall extracts obtained from exponentially growing
yeast-like cells was performed (De Groot et al., 2004). By use
of hydrolysis with hydrofluoric acid (HF)-pyridine, which
cleaves phosphodiester bonds, followed by liquid chromatography/mass spectrometry, 12 GPI-CWPs were identified.
Five of them (Cht2, Crh11, Pga4, Phr1 and Scw1) have
different enzymatic activities related to carbohydrates, two
are adhesive proteins (Als1 and Als4), one is related to
flocculins (Pga24) and another one appears to be a superoxide dismutase (Sod4/Pga), whereas the functions of the
other three (Ecm33.3, Rbt5 and Ssr1) are unknown. In the
pathogenic related species C. glabrata, similar proteomic
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analysis revealed the presence of two structural GPI proteins
(CWP1.1p and CWP1.2p) and Crh1p, a putative 1,3-bglucan remodelling enzyme (Weig et al., 2004).
Pir proteins
These are highly O-glycosylated proteins characterized by
the presence of internal repeats in variable number (Fig. 2;
Table 2) (Toh-e et al., 1993). Their presence in ascomycetes
and deuteromycetes appears to be universal, and their
organization is similar, including the presence of a signal
peptide, a Kex2 sensitive site, a domain with two to 11
repetitive sequences, and a C-terminal sequence with four
Cys residues at identical positions (repeat(s)-Cys–66aa–Cys–16aa–Cys–12aa–Cys–COOH). Pir proteins do not contain
a GPI anchor motif and are attached to the cell wall by
unknown alkali-labile bonds, possibly O-glycosidic linkages
with b-1,3-glucan. At least some of the Pir proteins are
retained in the wall exclusively by disulfide bridges, given
that some of them are released by reducing agents such as bME or DTT (Castillo et al., 2003; Toh-e et al., 1993). In C.
albicans, an antibody directed to the S. cerevisiae Pir protein
Hsp150 recognized two proteins extracted by alkali or b-1,3glucanase, and a high-molecular-mass protein secreted to
the growth medium, demonstrating the existence of Pirrelated proteins in the fungus (Kandasamy et al., 2000). In
similar experiments, Western blot analysis using an antiserum directed against S. cerevisiae Pir2p/Hsp150 revealed the
presence of at least two differentially expressed Pir2 homologs in the cell surface of C. albicans (Kapteyn et al., 2000). It
was also observed that in Dmnn9 and Dpmt1 mutants, which
are defective in N- or O-glycosylation, respectively, as well as
in a Dkre6 mutant, the amounts of Pir proteins were slightly
up-regulated (Kapteyn et al., 2000). Recent results have
revealed that C. albicans contains a single Pir-proteinencoding gene. By use of mass spectrometry and in silico
analyses, two Pir proteins encoded by nonidentical alleles of
a single gene (CaPIR1) were identified (Martinez et al.,
2004). Both encoded proteins contained a single N-mannosylated chain, four Cys residues and seven repeats, but one of
them was 21 amino acids shorter. Homozygous mutants
were impossible to obtain, suggesting that the gene is
essential for growth. Heterozygous mutants displayed an
abnormal phenotype associated with wall alterations (Martinez et al., 2004). In this regard, in a parallel study (De
Groot et al., 2004) similar analysis of NaOH-released
proteins led to the identification of two Pir proteins (named
Pir1 and Pga29). It is feasible that these correspond to the
two allelic products of CaPIR1 described above.
Atypical proteins
Regarding atypical wall proteins belonging to subgroup 2b
(see above and Table 2) in C. albicans, numerous examples
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exist. However, it is important not to confuse the two types:
(i) the highly glycosylated proteins that contain a signal
peptide and are secreted through the normal pathway
mostly to the medium, but retained in the wall in variable
proportions where they have different functions, and (ii)
those proteins that have been amply recognized to be of
cytoplasmic origin and to lack the characteristics of a bona
fide secreted protein, and that have been recently detected in
significant amounts in the cell wall. Only the latter are the
so-called ‘atypical proteins’.
Proteins related to the hsp70 and hsp90 families of
conserved stress proteins were found in the cell wall of C.
albicans, apparently as bona fide components (reviewed in
Chaffin et al., 1998). Higher levels of enolase and two
subforms of phosphoglyceromutase were detected in the
wall of a fluconazol-sensitive strain, as compared to a
resistant one, which displayed higher levels of two exoglucanases (Angiolella et al., 2002). Using a lgt11 cDNA library
from C. albicans to screen sera from patients with systemic
candidiasis, a cDNA has been identified corresponding to
the gene encoding 3 phosphoglycerate kinase (Alloush et al.,
1997). The protein is located at the surface of the wall
mainly of yeast cells, being released by treatment with b-ME.
A similar protocol using antibodies directed to cell wall
proteins led to the identification of C. albicans genes
encoding several nontypical wall proteins, which were
located in the cell wall later on by standard protocols. One
of these genes encoded 3-phosphoglycerate kinase, and
another one was a novel gene encoding two products: a
cytochrome c haem lyase targeted to the mitochondria, and
a cell wall protein (Cervera et al., 1998). Whether these
proteins are adventitiously trapped in the cell wall, as has
been suggested by some authors, or somehow are actively
released and associate to the wall polymers, as suggested by
others (see above), is still a matter of discussion. Until a
decisive answer is obtained, it is advisable to treat this matter
cautiously.
Nature of the carbohydrate moieties of wall
proteins
Different forms of glycosylation have been described in
relation to wall proteins: N-glycosylation, O-glycosylation,
and attachment of a GPI anchor. Normally, these proteins
are rich in Ser and Thr residues where O-glycosylation
occurs, and contain the sequons, tripeptides of N-Asn-XaaSer/Thr, where N-glycosylation takes place. The level of
glycosylation is variable, often as high as 50–95% by weight.
Branched oligosaccharide moieties of high molecular weight
made mostly of mannosyl units are connected to the peptide
by N-glycosidic bonds between N-acetylchitobiosyl and
asparagine moieties. Two distinct parts have been detected
in the glycosidic moiety of glycoproteins, an inner core of
FEMS Yeast Res 6 (2006) 14–29
23
Molecular organization of the cell wall of Candida albicans
10–14 residues (Asn-GlcNAc2-Man10–14), a large outer chain
containing a backbone of a-1,6-linked mannose residues,
and a variable number of a-1,2-linked mannose side chains.
The side chains in turn contain a variable number of a-1,2
and a-1,3-bound mannosyl moieties, and occasionally phosphomannosyl units that confer a net negative charge on the
cell wall, which contributes to the properties of the cell
surface.
Regarding O-glycosylated proteins, chains of up to five a1,2 and a-1,3-bound mannose units are linked to Ser or Thr
residues. This bond is sensitive to weak alkali treatment (belimination). These proteins are characterized by their rigid
structure that protrudes from the wall surface in a manner
similar to antennae (for a review, see Orlean, 1997).
In relation to the carbohydrate moieties of the wall
proteins of C. albicans, special mention should be made to
a lipid-modified polysaccharide called phospholipomannan,
which is a modified member of the MIPC family, but due to
its hydrophilic properties given by the large polymannose
moiety it locates in the cell wall. This molecule seems to be
the center of the host cell response following deep C.
albicans infections, but the mechanisms for this have not
yet been elucidated (Jouault et al., 2003).
Roles of wall proteins
Wall proteins are absolutely essential for the life of C.
albicans, as with all fungi. Proteins have extremely important and varied roles, among which we can cite the following
five.
Enzymatic
Some wall enzymes play a role in the degradation of large
impermeable molecules, making the products accessible for
cell nutrition; others are involved in the degradation of cell
wall polymers or in their synthesis, being necessary for wall
and therefore for cell growth. Accordingly, several degradative enzymes have been found located in the wall of C.
albicans (Chaffin et al., 1998; Pedreño et al., 2004). Also, a
number of GPI proteins display enzymatic activities related
to the synthesis and modulation of wall components (chitinases, glucanases, etc.).
Cell interaction
Other wall proteins are involved in the interaction with
other cells. Glycoproteins present in fimbriae take an active
role in this sense. Fimbriae are external protruding filamentous structures that provide the initial contact of pathogenic
fungi such as C. albicans with host cells (Tokunaga et al.,
1990). Fimbriae mannoproteins are made of 80–85% DFEMS Yeast Res 6 (2006) 14–29
mannose and 10–15% protein. Among other surface proteins involved in interactions with other cells, or inert
substrates, we may cite wall proteins involved in adhesion
of C. albicans to host tissues and some of their products such
as fibrinogen, complement fragments and several extracellular matrix components. Attachment and adherence of C.
albicans has been described to depend on at least four
recognition systems, classified according to the type of
adhesins, the kind of host cells (epithelial, endothelial or
platelets) and the type of host cell ligand (carbohydrate or
protein) (for a review, see Calderone, 1993). System I
adhesin corresponds to a mannoprotein with lectin-like
properties that recognizes fucosyl or glucosaminyl glycosides of epithelial cells. System II adhesin functionally
resembles the ‘integrin’ receptors of mammalian cells. It is
also a mannoprotein, but it recognizes proteins with the
RGD ligand of platelets, endothelial cells or RGD (Arg-GlyAsp acid) domains of extracellular matrix proteins of
endothelial cells. System III adhesin (mannan) promotes
the adherence of the fungus to epithelial cells. However, this
adhesin clearly differs from the system I adhesin, and it
utilizes the protein component to recognize the ligand.
System IV adhesin (mannoprotein) seems to be associated
with the colonization of splenic tissue by the pathogen. The
C. albicans ALS gene family is composed of eight genes
(ALS1 to ALS7 and ALS9) encoding cell wall glycoproteins
involved in adhesion to host surfaces. Allelic variations in
some of these genes suggest that they may be related to the
evolutionary stress to which the fungus has been subjected.
Analysis of the structure of these proteins, their cloning and
expression in S. cerevisiae cells, and analysis of the adherence
characteristics of the transgenic yeasts have provided evidence of the extreme capacity of adaptation in C. albicans
(Sheppard et al., 2004).
Antigenicity
Different wall proteins are antigenic, as occurs with several C.
albicans glycoproteins that are differentially present in
the yeast or mycelial forms (Sundstrom et al., 1988). A
47-kDa protein component of different strains of C. albicans
was identified as an immunodominant antigen present in the
cytoplasm and cell wall of both yeast and mycelial cells, but
mainly exposed on the outer surface and unable to bind to
Concanavalin A (Matthews et al., 1988). An interesting wall
glycoprotein of 260 kDa, specific to the mycelial form of C.
albicans, was isolated and monoclonal antibodies raised
against its polypeptide moiety (Casanova et al., 1989). (It is
relevant to recall that antigenicity of different wall glycoproteins has been associated not with the protein but with their
mannan moiety.) Interestingly, Fab fragments from these
monoclonal antibodies inhibited the yeast-to-mycelium dimorphic transition of the fungus (Casanova et al., 1990).
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24
Pathogenicity
An important role of some wall proteins is their involvement
in the establishment of the pathogen in the host, or in the
response of the latter to invasion. The notion that in
pathogenic fungi wall proteins frequently constitute virulence factors is widely accepted. In relation to this, it is
known that the wall proteins extracted by b-ME exacerbate
collagen-induced arthritis in mice, and, as described above,
a number of mutants deficient in proteins located or related
to cell wall construction display reduced virulence. This type
of information provides evidence on the role of wall proteins
as pathogenicity or virulence factors, either directly or
indirectly (Fradin et al., 2005)
Wall structure and morphogenesis
Finally, other glycoproteins may be important from a
structural point of view, and for the morphogenetic response of the fungus. An example of this is a putative surface
glycosidase (Csf4), identified as an important factor for cell
wall integrity and maintenance. Deletion of CSF4 reduced
hyphal growth, adherence to mammalian cells and virulence
in a mouse model (Alberti-Segui et al., 2004). A further
example is the 260-kDa protein that, as described above, was
essential for mycelial growth. GPI protein Dfg5p has also
been found to be involved in dimorphism. Mutants lacking
this protein are defective in hypha formation at alkaline pH,
suggesting that Dfg5p is involved in the transmission of the
surface signal required for hyphal growth (Casanova et al.,
1990). Mutants defective in another GPI protein,
CaEcm33p, display morphological alterations, including
reduced hyphal formation, defects in wall structure and
reduced virulence in a murine model (Martinez-Lopez
et al., 2004). Finally, we must recall the CaPir1 protein cited
above, which is apparently required for the correct formation of the cell wall, because null mutants are inviable
(Martinez et al., 2004).
Cell wall maturation and organization
It is not an exaggeration to suggest that organization of the
cell wall components is initiated at the level of transcription.
In Saccharomyces cerevisiae, two transcription factors, Ace2p
and Swi5p, are key regulators of cell wall synthesis. In
agreement with these results, mutation of the gene CaACE2,
which is the sole homolog of these genes in C. albicans,
provoked severe alterations in functions that depend on the
normal structure of the cell wall (Kelly et al., 2004). Mutant
cells displayed defects in cell separation, hyphal growth,
adhesion and virulence in a mouse model, suggesting that
cell wall organization depended on the orchestrated synthesis, controlled by the transcription factor encoded by
CaACE2, of a number of cell products.
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It is important to recall that the cell wall is a coherent
structure. Accordingly, in order to achieve its final structure,
the establishment of connections among its different components becomes necessary. Some of these associations
involve hydrogen, hydrophobic or polar bonding, but others
take place through covalent linkages. These associations
must occur in the cell wall itself, where they produce changes
in its mechanical properties (for a review, see Ruiz-Herrera
et al., 2004). The Family 72 of transglycosidases may
function in the cross-linking of b-glucans in the cell wall
(Fonzi, 1999; Mouyna et al., 2000). Also a transglycosylation
reaction catalyzed by a wall-located b-1,3-glucanase was
reported to occur in C. albicans, apparently being involved
in branching of b-1,3-glucan (Goldman et al., 1995). In
vitro, the enzyme cleaved laminaribiose from the reducing
end of a linear b-1,3-glucan and transferred the remainder
to another laminarioligosaccharide, to give a product containing a b-1,3-b-1,6-branchpoint (Goldman et al., 1995).
Mutation of BGL2, the gene encoding this b-1,3 glucosyltransferase in a clinical strain of C. albicans, gave rise to
reduction in growth rate, cell aggregation during stationary
phase, decrease in virulence in a murine model and increase
in sensitivity to nikkomycin Z (Sarthy et al., 1997). The
observation that null mutants still displayed b-1,3-glucosyltransferase activity made the authors suggest the presence of
additional enzymes exercising the same activity. As described above, b-1,3-glucan and chitin in C. albicans (Surarit
et al., 1988) are covalently linked, and the formation of such
a bond must occur in the wall itself. Another type of
association of wall macromolecules takes place among
proteins. This may occur through the formation of disulfide
bonds, a redox reaction which is not fully understood,
whereas other reactions may account for the association
between O-linked and N-linked mannoproteins. Accordingly, the covalent bonding of an O-glycosylated epitope to
N-glycosylated mannoproteins was reported to occur in the
cell wall of C. albicans (Elorza et al., 1989). Synthesis of the
cell wall in fungi is under the control of mechanisms that
monitor its integrity and characteristic resistance. In S.
cerevisiae, a decrease in the synthesis of some wall polysaccharides gives rise to a significant increase in the levels of
chitin. For example, mutants deficient in O-glycosylation
(pmt mutants) or N-glycosylation (mnn mutants) contained
twice the normal levels of chitin (Gentzsch & Tanner, 1996).
These mutants also contained higher amounts of Pir and
GPI proteins (Kapteyn et al., 2000). Mutants of C. albicans
and C. glabrata deficient in b-1,6-glucan synthesis also had
increased levels of chitin in the wall. It has been suggested
that these alterations in the chemical composition of the
wall may operate as compensatory mechanisms to guarantee
its integrity. Their mode of action involves mitogen activated protein kinase (MAPK) cascades, as occurs with other
signaling pathways. In C. albicans, a MAPK named Mkc1p
FEMS Yeast Res 6 (2006) 14–29
25
Molecular organization of the cell wall of Candida albicans
has been identified as a homolog of Mpk1p (Slt2) from S.
cerevisiae (Navarro-Garcia et al., 1998), the MAPK involved
in the regulation of cell wall integrity. Homozygous mkc1
mutants showed alterations in the cell surface, evidenced by
scanning electron microscopy, increased amounts of specific
cell wall epitopes, and higher sensitivity to inhibitors of bglucan and chitin synthesis. Using the two-hybrid system, it
was demonstrated that Mkc1p was able to interact specifically with yeast Mkk1p and Mkk2p, the MAPK kinases of the
Pkc1p-mediated route. MAPK may not be the sole pathway
involved in the regulation of cell wall integrity. Recent data
have provided evidence that the phenomenon also involves a
signaling pathway dependent on phosphoinositides (Schorr
et al., 2001).
With regard to the sensing mechanisms, it seems relevant
that at least three putative two-component histidine kinase
signal transduction proteins, including Chk1p and a response regulator protein (Cssk1p), exist in C. albicans. To
determine the role of these systems, chk1 mutants have been
obtained. These became avirulent in a murine model, and
displayed significant changes related to cell wall composition and structure. Accordingly, the alkali-soluble carbohydrate levels, as well as the Mr of acid-stable mannan species,
were reduced, the O-linked oligosaccharides appeared truncated, and the degree of polymerization and the ratio of b1,3/b1,6 linkages in b-glucans were all reduced (Kruppa
et al., 2003). In further analyses, immunoelectron microscopy with specific antibodies directed to surface isotopes
revealed alterations of the cell surface, not only in chk1
mutants but also in mutants defective in the other two genes
encoding histidine kinases (HK) of the fungus (SLN1 and
NIK1). Analysis of the expression of 29 genes involved in
mannan biosynthesis revealed similar alterations in all
mutants (Kruppa et al., 2004). All these data have been
taken as evidence for a role of the two-component histidine
kinase signal transduction proteins in the regulation of cell
wall structure in C. albicans. Finally, it is also significant that
the repair mechanisms induced by the perturbations in the
cell wall construction of S. cerevisiae mutants (fks1, Kre6,
mnn9, gas1 and knr4) integrate three major regulatory
systems: the PKC1-SLT2 MAPK signaling unit, the Ca21/
calcineurin-dependent signaling conduct and the general
stress system (Lagorce et al., 2003). Recently, a global model
integrating different pathways and processes in relation with
the fungal status (commensal or pathogenic) has been
proposed by the European Consortium ‘Novel approaches
for the control of fungal diseases’ (Fig. 3).
Fig. 3. Global model interconnecting Candida albicans as a commensal
organism and its pathogenic status as a function of the host immune
situation. In an immunocompentent host, the microorganism is found
only as a commensal organism. In the immunocompromised host, the
situation is different because the passive defenses are unable to block
virulence factors such as cell wall remodeling, biofilm formation, metabolic reprogramming, etc., resulting in its colonization (Brown et al.,
2003).
introduction of different modern techniques has allowed
comparative analyses of the structural components, and in
silico analyses of the genome and sequencing of the enzymatic and structural proteins have deepened our knowledge
on how the cell wall is organized. Comparison of the results
uncovered in C. albicans with those obtained with other
fungi has demonstrated the similarities in the mechanisms
of cell wall synthesis and construction in different fungi, but
also the existence of important differences that reveal the
uniqueness of the C. albicans species. Among the important
differences disclosed, we may cite the structure of b-1,3glucan and Pir proteins, the biosynthetic mechanism of b1,6-glucans, the different number and roles of chitin
synthases and Pir proteins, and the antigenic and adhesive
characteristics of the cell wall surface. Also, the observation
that some components of the C. albicans cell wall play
important roles in pathogenicity and virulence imposes a
strong distinction between the cell wall of this species and
that of their saprophytic relatives. Finally, it should be
stressed that our extended knowledge of the role of wall
components in the pathogenic behavior of C. albicans is
leading to a better comprehension of the molecular bases of
the invasiveness and aggressiveness of the fungus, and
accordingly to the design of better strategies for its control.
Acknowledgements
Final considerations
From the data presented above, it is evident that our
concepts on the structure, synthesis and organization of the
cell wall of Candida albicans have rapidly evolved. The
FEMS Yeast Res 6 (2006) 14–29
Original work from the laboratories of J.R.H., R.S., E.V. and
M.V.E. was supported, respectively, by CONACYT, Mexico,
and the European Union (QLK2CT-2000-0795 and MRTNCT-2003-504148), Spanish Ministerio de Ciencia y Cultura
2005 Federation of European Microbiological Societies
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26
(BMC2003-01023) and Agència Valenciana de Ciència i
Tecnologia de la Generalitat Valenciana (Grupos 03/187).
We acknowledge the helpful discussions with our colleagues
in the EC Galar Fungail Consortium.
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